Penatti et al. Arthritis Research & Therapy (2017) 19:103 DOI 10.1186/s13075-017-1305-1
RESEARCH ARTICLE
Open Access
Differences in serum and synovial CD4+ T cells and cytokine profiles to stratify patients with inflammatory osteoarthritis and rheumatoid arthritis Alessandra Penatti1,3*†, Federica Facciotti2*† , Roberta De Matteis5, Paola Larghi2,6, Moira Paroni2, Antonella Murgo3, Orazio De Lucia3, Massimiliano Pagani2, Luca Pierannunzii3, Marcello Truzzi3, Andreea Ioan-Facsinay4, Sergio Abrignani1,2, Jens Geginat2† and Pier Luigi Meroni1,3,5†
Abstract Background: The aim was to investigate CD4+T-cell subsets, immune cells and their cytokine profiles in blood and synovial compartments in rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) to define specific immune signatures. Methods: Peripheral blood, synovial fluid (SF) and synovial membranes (SM) of RA and OA patients were analyzed. CD4+T-cell subset frequencies were determined by flow cytometry, and cytokine concentrations in serum and SF were measured by ELISA. Results: In peripheral blood, OA patients had altered frequencies of regulatory T-cell subsets, and higher frequencies of Th17 and of Th1/17 cells than RA patients. In the synovial compartment of OA patients, conventional Th17 cells were largely excluded, while Th1/17 cells were enriched and more frequent than in RA patients. Conversely, in the synovial compartment of RA patients, regulatory T cells and Tfh cells were enriched and more frequent then in OA patients. IL-17 and Blys were increased both in serum and SF of RA patients, and correlated with autoantibodies and disease activity. Notably, Blys levels were already significantly elevated in RA patients with low disease activity score in 28 joints (DAS28) and without autoantibody positivity. Conclusions: Although patients with inflammatory OA have immune activation in the synovial compartment, they display different T-cell subset frequencies and cytokine profiles. Soluble mediators such as Blys might help to discriminate mild clinical forms of RA from inflammatory OA particularly at the onset of the disease. Keywords: Rheumatoid arthritis, Inflammatory osteoarthritis, T helper subsets, Cytokines, Blys
Background Rheumatoid arthritis (RA) and osteoarthritis (OA) represent two manifestations of inflammatory arthritides. Although the etiopathology of these diseases is different, they both involve synovial inflammation [1]. While * Correspondence: [email protected]; [email protected]; [email protected] † Equal contributors 1 DISCCO-Department of Clinical Science and Community Health Università degli Studi di Milano, 20122 Milan, Italy 2 INGM-National Institute of Molecular Genetics “Romeo ed Enrica Invernizzi”, 20122 Milan, Italy Full list of author information is available at the end of the article
RA is an autoimmune disease characterized by chronic relapsing-remitting inflammation of the peripheral joints [2], OA is induced by multifactorial mechanisms established primarily by biomechanical stress [3]. Thus, for a long time it has been believed that only patients with RA may have immune cells in the synovial infiltrate. However, recent reports showed that patients with OA also have inflammatory synovial infiltrates that, particularly in the most severe forms, are characterized by high-grade inflammation and could lead to abrupt onset, marked clinical symptoms and structural damage [4]. Those patients with inflammatory OA have inflammatory features that in some
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Penatti et al. Arthritis Research & Therapy (2017) 19:103
Page 2 of 9
instances resemble those of RA. CD4+T-cells are key to the initiation and progression of synovitis [2, 5] in patients with RA, and cytokines released into the synovial fluid (SF) by the inflamed tissue likely reflect the composition of effector and regulatory T cells infiltrating the synovia [6]. Relatively little is known about the characteristics of synovial immune cells in patients with inflammatory OA, although recent studies suggest that CD4 + T cells might contribute to the pathophysiology of OA and correlated symptoms, such as pain [7, 8]. Here, we further evaluated the role of CD4+ T cells by in-depth analysis of subset composition and the cytokine milieu in peripheral blood and SF during effusion episodes in patients with RA and OA, and of the synovial membrane in patients who also underwent joint biopsy or surgery. We tested correlation between the disease activity parameters and the immunologic alterations in order to determine whether different synovial profiles of immune cells or cytokines could be specifically associated with different types of arthropathy. We report that patients with inflammatory OA display synovial immune activation that presents different signatures in comparison to RA.
Methods Human samples and patients
Buffy-coat blood from 25 healthy age-matched and sexmatched donors (mean age 42 years; age range 26–58
years; male/female ratio 10/15) were obtained from the IRCCS Policlinico Ospedale Maggiore, Milan, Italy. There were 25 patients with RA (mean age 56 years; age range 40–72 years; male/female ratio 12/13) and 18 patients with inflammatory OA (mean age 69 years; age range 57–86 years; male/female ratio 8/10), who were attending the Rheumatology Department of the ASST-Gaetano Pini/ CTO Orthopedic and Traumatology Specialist Center of Milan, enrolled in this study. All patients with RA fulfilled the European League Against Rheumatism/American College of Rheumatologists (EULAR/ACR) 2010 classification criteria and their disease activity were assessed using the disease activity score in 28 joints (DAS28) based on the erythrocyte sedimentation rate (ESR) [9]. The median duration of RA was 4.3 ± 3.9 years. We included symptomatic patients with radiographic evidence of mild knee OA (Kellgren and Lawrence grade 2) with an active inflammatory phenotype characterized by the presence of knee effusion synovitis and synovial thickening detected by ultrasound (US) [10]. Clinical, biochemical and cellular data on the enrolled patients with RA and OA are summarized in Table 1. Peripheral blood (PB) was obtained and collected in heparinized tubes. SF samples from the knee joints of patients with RA and OA were collected during therapeutic needle aspiration into heparinized tubes. SM samples were taken from two patients with RA who also underwent knee
Table 1 Summary of clinical, biochemical and cellular data on patients with RA and OA Characteristic
Rheumatoid arthritis
Osteoarthritis
PBa
SFb
SMc
PBa
SFb
SMc
25
9
4
11
10
9
RF (n)
17
9
4
ACPA (n)e
17
9
4
-
-
-
Number d
f
DAS28 index, low (0–3) mean 2.42
10/25
0/9
3/4
-
-
-
DAS28f index, medium (3–5) mean 4.26
12/25
6/9
1/4
-
-
-
DAS28f index, high (>5) mean 5.55
3/25
3/9
0/4
-
-
-
ESRg (mm/h)
56.3 (5–87)
10.3 (1–25)
CRPh (mg/dl)
4.3 (0.4–7.5)
0.27 (0–0.6)
i
NSAID (+/−)
8/25
14/18
Corticosteroids (+/−)
14/25
0/18
DMARDsj (+/−)
25/25
0/18
PBMCsk (106/ml)
1.8 (1.4–2.3)
l
1.6 (1.3–1.9) 5
SFMCs
9.2 (4.6–18.2) *104/ml
6.2 (3.6–11.3) *10 /ml
SMMCsm
3.2 *106
1.3 *106
PB peripheral blood, SF synovial fluid, SM synovial membrane, RF rheumatoid factor, ACPA anti-citrullinated protein antibody, fDAS28 disease activity score based on 28 joint counts, gESR erythrocyte sedimentation rate, hCRP C-reactive protein (CRP), iNSAIDs non-steroidal anti-inflammatory drugs, jDMARDs diseasemodifying anti-rheumatic drugs, kPBMCs peripheral blood mononuclear cells, lSFMCs synovial fluid mononuclear cells, mSMMCs synovial membrane mononuclear cells. *Mean, (values range). dRF nephelometry assay, cutoff value >15 UI/ml. All patients with RA had high positive dRF levels (more than three times the ULN for the laboratory and assay): 264 ± 190. eACPA: QUANTA Lite® CCP3.1 IgG/IgA ELISA (INOVA Diagnostics), cutoff value >20 UI/ml. All patients with RA had high positive eACPA levels (more than three times the ULN for the laboratory and assay):125 ± 64. hCRP cutoff value 1 mg/dl. a
b
c
d
e
Penatti et al. Arthritis Research & Therapy (2017) 19:103
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joint biopsy and from nine patients with OA and two patients with RA who additionally had knee replacement surgery. The majority of patients with RA had medium/low disease activity (as summarized in Table 1), and were on medium/low doses of disease modifying anti-rheumatic drugs (DMARDs) (hydroxychloroquine, methotrexate or leflunomide). Thirteen out of twenty-four patients were on low-dose corticosteroid therapy ( 0.05 for all. (TIF 642 kb) Additional file 8: Figure S8. Distribution of T helper (a, c) and T regulatory (b, d) subsets in peripheral blood (upper panels) and synovial fluid (lower panels) of significantly different subsets in patients with RA according to CS therapy: statistical analysis by Mann-Whitney test, p > 0.05. Peripheral blood: (a) Th1/17, Th17; (b) Tr1 and TReg subsets. Synovial fluid: (c) Th1/17; (d) TReg subsets. (TIF 607 kb)
Abbreviations ACPA: Anti-citrullinated protein antibody; DAS29: disease activity score in 28 joints; DMARDs: Disease-modifying anti-rheumatic drugs; ELISA: Enzyme-linked immunosorbent assay; ESR: erythrocyte sedimentation rate; HD: Healthy donors; IL: interleukin; NK: Natural killer; NSAIDs: non-steroidal anti-inflammatory drugs; OA: Osteoarthritis; PB: Peripheral blood; PBMCs: Peripheral blood mononuclear cells; RA: Rheumatoid arthritis; RF: Rheumatoid factor; SF: Synovial fluid; SFMCs: Synovial fluid mononuclear cells; SM: Synovial membranes; SMMCs: Synovial membrane mononuclear cells; Th: T helper; Tregs: T regulatory cells; US: Ultrasound Acknowledgements Not applicable. Funding This work was supported by the Cariplo foundation, by the Romeo ed Enrica Invernizzi foundation and by Ricerca Corrente 2015–16 IRCCS Istituto Auxologico Italiano. Availability of data and materials Supporting information for Figures S1, S2, S3, S4, S5, S6, S7 and S8 are available online. Authors’ contributions AP participated in the study design, obtained data, participated in the statistical analysis and drafted the manuscript; FF participated in the study design and coordination, obtained data, participated in the statistical analysis and wrote the manuscript; RDM and PL performed experiments; MP participated in the analysis of the data and in the drafting of the manuscript; AM and ODL recruited patients with OA and RA and obtained biological samples; LP and MT obtained biological samples; SA, MP and AIF participated in the design of the study, in the interpretation of the data and in the drafting of the manuscript; JG and PLM conceived the study, participated in its design and coordination, in analysis and interpretation of data and in the drafting of the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethical approval and consent to participate The ethical committee (Milano Area B) approved the use of blood samples and synovium specimens for research purposes (permission 2476 Comitato Etico Milano Area B/Parere favorevole condizionato datato 02.10.2014/parere 591bis") and informed consent was obtained from all patients. Source of support None declared.
Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 DISCCO-Department of Clinical Science and Community Health Università degli Studi di Milano, 20122 Milan, Italy. 2INGM-National Institute of Molecular Genetics “Romeo ed Enrica Invernizzi”, 20122 Milan, Italy. 3 ASST-Gaetano Pini/CTO Orthopedic and Traumatology Specialist Center, Rheumatology and Orthopedic Department, 20122 Milan, Italy. 4Department of Rheumatology, Leiden University Medical Center, 2300 Leiden, The Netherlands. 5Laboratory of immuno-rheumatological researches, IRCCS Istituto Auxologico Italiano, 20149 Milan, Italy. 6Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20122 Milan, Italy.
Penatti et al. Arthritis Research & Therapy (2017) 19:103
Page 9 of 9
Received: 1 December 2016 Accepted: 2 May 2017
References 1. Beaten D, et al. Comparative study of the synovial histology in rheumatoid arthritis, spondyloarthropathy, and osteoarthritis: influence of disease duration and activity. Ann Rheum Dis. 2000;59:945–53. 2. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. NEJM. 2011; 365:2205–19. 3. Hunter DJ. Osteoarthritis. Best Pract Res Clin Rheumatol. 2011;25:801–14. 4. Sellam J, Berenbaum F. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis. Nat Rev Rheumatol. 2010;6:625–35. 5. Mellado M, et al. T cell migration in rheumatoid arthritis. Front Immunol. 2015;6:384. 6. McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol. 2007;7:429–42. 7. Lange-Brokaar BJE, et al. Synovial inflammation, immune cells and their cytokines in osteoarthritis: a review. Osteoarthritis Cartilage. 2012;20:1484–99. 8. Klein-Wieringa IR, et al. Inflammatory cells in patients with endstage knee osteoarthritis: a comparison between the synovium and the infrapatellar fat pad. J Rheumatol. 2016;43(4):771–8. 9. Aletaha D, et al. Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 2010;62:2569–81. 10. Hart DJ, Spector TD. The classification and assessment of osteoarthritis. Baillieres Clin Rheumatol. 1995;9(2):407–32. 11. Geginat J, Paroni M, Facciotti F, Gruarin P, Kastirr I, Caprioli F, Pagani M, Abrignani S. The CD4-centered universe of human T cell subsets. Semin Immunol. 2013;25(4):252–62. Epub 2013 Nov 1. 12. Morita R, et al. Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity. 2011;34:108–21. 13. Acosta-Rodriguez EV, Rivino L, Geginat J, et al. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol. 2007;8:639–46. 14. Paroni M, Maltese V, De Simone M, Ranzani V, Larghi P, et al. Recognition of viral and self-antigens by Th1 and Th1/Th17 central memory cells in patients with multiple sclerosis reveals distinct roles in immune surveillance and relapses. J Allergy Clin Immunol. 2017 [Epub ahead of print]. 15. Facciotti F, Gagliani N, Häringer B, Alfen JS, Penatti A, et al. IL-10-producing forkhead box protein 3-negative regulatory T cells inhibit B-cell responses and are involved in systemic lupus erythematosus. J Allergy Clin Immunol. 2016;137(1):318–21. 16. Yamada H, et al. Preferential accumulation of activated Th1 cells not only in rheumatoid arthritis but also in osteoarthritis joints. J Rheumatol. 2011;38:1569–75. 17. Nistala K, et al. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proc Natl Acad Sci USA. 2010;107:14751–6. 18. Paulissen SMJ, et al. The role and modulation of CCR6+ Th17 cell populations in rheumatoid arthritis. Cytokine. 2015;74:43–53. 19. Paulissen SMJ, et al. CCR6+ Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis. Arthritis Res Ther. 2015;17:344. 20. Cheema GS, et al. Elevated serum B Lymphocyte stimulator levels in patients with systemic immune–based rheumatic diseases. Arthritis Rheum. 2001;44(6):1313–9. 21. Wei F, et al. The role of BAFF in the progression of rheumatoid arthritis. Cytokine. 2015;76:537–44. 22. Geginat J, et al. The light and the dark sides of Interleukin-10 in immunemediated diseases and cancer. Cytokine Growth Factor Rev. 2016;30:87–93. 23. Moradi B, et al. CD4 + CD25 + hiCd127lw regulatory T cells are enriched in rheumatoid arthritis and ostheoarthritis joints-analysis of frequency and phenotype in synovial membrane, synovial fluid and peripheral blood. Arthritis Res Ther. 2014;16:R97. 24. Appel H, et al. An elevated level of IL-10- and TGF β-secreting T cells, B cells and macrophages in the synovial membrane of patients with reactive arthritis compared to rheumatoid arthritis. Clin Rheumatol. 2004;23:435–40.
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RESEARCH ARTICLE
Open Access
Differences in serum and synovial CD4+ T cells and cytokine profiles to stratify patients with inflammatory osteoarthritis and rheumatoid arthritis Alessandra Penatti1,3*†, Federica Facciotti2*† , Roberta De Matteis5, Paola Larghi2,6, Moira Paroni2, Antonella Murgo3, Orazio De Lucia3, Massimiliano Pagani2, Luca Pierannunzii3, Marcello Truzzi3, Andreea Ioan-Facsinay4, Sergio Abrignani1,2, Jens Geginat2† and Pier Luigi Meroni1,3,5†
Abstract Background: The aim was to investigate CD4+T-cell subsets, immune cells and their cytokine profiles in blood and synovial compartments in rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) to define specific immune signatures. Methods: Peripheral blood, synovial fluid (SF) and synovial membranes (SM) of RA and OA patients were analyzed. CD4+T-cell subset frequencies were determined by flow cytometry, and cytokine concentrations in serum and SF were measured by ELISA. Results: In peripheral blood, OA patients had altered frequencies of regulatory T-cell subsets, and higher frequencies of Th17 and of Th1/17 cells than RA patients. In the synovial compartment of OA patients, conventional Th17 cells were largely excluded, while Th1/17 cells were enriched and more frequent than in RA patients. Conversely, in the synovial compartment of RA patients, regulatory T cells and Tfh cells were enriched and more frequent then in OA patients. IL-17 and Blys were increased both in serum and SF of RA patients, and correlated with autoantibodies and disease activity. Notably, Blys levels were already significantly elevated in RA patients with low disease activity score in 28 joints (DAS28) and without autoantibody positivity. Conclusions: Although patients with inflammatory OA have immune activation in the synovial compartment, they display different T-cell subset frequencies and cytokine profiles. Soluble mediators such as Blys might help to discriminate mild clinical forms of RA from inflammatory OA particularly at the onset of the disease. Keywords: Rheumatoid arthritis, Inflammatory osteoarthritis, T helper subsets, Cytokines, Blys
Background Rheumatoid arthritis (RA) and osteoarthritis (OA) represent two manifestations of inflammatory arthritides. Although the etiopathology of these diseases is different, they both involve synovial inflammation [1]. While * Correspondence: [email protected]; [email protected]; [email protected] † Equal contributors 1 DISCCO-Department of Clinical Science and Community Health Università degli Studi di Milano, 20122 Milan, Italy 2 INGM-National Institute of Molecular Genetics “Romeo ed Enrica Invernizzi”, 20122 Milan, Italy Full list of author information is available at the end of the article
RA is an autoimmune disease characterized by chronic relapsing-remitting inflammation of the peripheral joints [2], OA is induced by multifactorial mechanisms established primarily by biomechanical stress [3]. Thus, for a long time it has been believed that only patients with RA may have immune cells in the synovial infiltrate. However, recent reports showed that patients with OA also have inflammatory synovial infiltrates that, particularly in the most severe forms, are characterized by high-grade inflammation and could lead to abrupt onset, marked clinical symptoms and structural damage [4]. Those patients with inflammatory OA have inflammatory features that in some
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Penatti et al. Arthritis Research & Therapy (2017) 19:103
Page 2 of 9
instances resemble those of RA. CD4+T-cells are key to the initiation and progression of synovitis [2, 5] in patients with RA, and cytokines released into the synovial fluid (SF) by the inflamed tissue likely reflect the composition of effector and regulatory T cells infiltrating the synovia [6]. Relatively little is known about the characteristics of synovial immune cells in patients with inflammatory OA, although recent studies suggest that CD4 + T cells might contribute to the pathophysiology of OA and correlated symptoms, such as pain [7, 8]. Here, we further evaluated the role of CD4+ T cells by in-depth analysis of subset composition and the cytokine milieu in peripheral blood and SF during effusion episodes in patients with RA and OA, and of the synovial membrane in patients who also underwent joint biopsy or surgery. We tested correlation between the disease activity parameters and the immunologic alterations in order to determine whether different synovial profiles of immune cells or cytokines could be specifically associated with different types of arthropathy. We report that patients with inflammatory OA display synovial immune activation that presents different signatures in comparison to RA.
Methods Human samples and patients
Buffy-coat blood from 25 healthy age-matched and sexmatched donors (mean age 42 years; age range 26–58
years; male/female ratio 10/15) were obtained from the IRCCS Policlinico Ospedale Maggiore, Milan, Italy. There were 25 patients with RA (mean age 56 years; age range 40–72 years; male/female ratio 12/13) and 18 patients with inflammatory OA (mean age 69 years; age range 57–86 years; male/female ratio 8/10), who were attending the Rheumatology Department of the ASST-Gaetano Pini/ CTO Orthopedic and Traumatology Specialist Center of Milan, enrolled in this study. All patients with RA fulfilled the European League Against Rheumatism/American College of Rheumatologists (EULAR/ACR) 2010 classification criteria and their disease activity were assessed using the disease activity score in 28 joints (DAS28) based on the erythrocyte sedimentation rate (ESR) [9]. The median duration of RA was 4.3 ± 3.9 years. We included symptomatic patients with radiographic evidence of mild knee OA (Kellgren and Lawrence grade 2) with an active inflammatory phenotype characterized by the presence of knee effusion synovitis and synovial thickening detected by ultrasound (US) [10]. Clinical, biochemical and cellular data on the enrolled patients with RA and OA are summarized in Table 1. Peripheral blood (PB) was obtained and collected in heparinized tubes. SF samples from the knee joints of patients with RA and OA were collected during therapeutic needle aspiration into heparinized tubes. SM samples were taken from two patients with RA who also underwent knee
Table 1 Summary of clinical, biochemical and cellular data on patients with RA and OA Characteristic
Rheumatoid arthritis
Osteoarthritis
PBa
SFb
SMc
PBa
SFb
SMc
25
9
4
11
10
9
RF (n)
17
9
4
ACPA (n)e
17
9
4
-
-
-
Number d
f
DAS28 index, low (0–3) mean 2.42
10/25
0/9
3/4
-
-
-
DAS28f index, medium (3–5) mean 4.26
12/25
6/9
1/4
-
-
-
DAS28f index, high (>5) mean 5.55
3/25
3/9
0/4
-
-
-
ESRg (mm/h)
56.3 (5–87)
10.3 (1–25)
CRPh (mg/dl)
4.3 (0.4–7.5)
0.27 (0–0.6)
i
NSAID (+/−)
8/25
14/18
Corticosteroids (+/−)
14/25
0/18
DMARDsj (+/−)
25/25
0/18
PBMCsk (106/ml)
1.8 (1.4–2.3)
l
1.6 (1.3–1.9) 5
SFMCs
9.2 (4.6–18.2) *104/ml
6.2 (3.6–11.3) *10 /ml
SMMCsm
3.2 *106
1.3 *106
PB peripheral blood, SF synovial fluid, SM synovial membrane, RF rheumatoid factor, ACPA anti-citrullinated protein antibody, fDAS28 disease activity score based on 28 joint counts, gESR erythrocyte sedimentation rate, hCRP C-reactive protein (CRP), iNSAIDs non-steroidal anti-inflammatory drugs, jDMARDs diseasemodifying anti-rheumatic drugs, kPBMCs peripheral blood mononuclear cells, lSFMCs synovial fluid mononuclear cells, mSMMCs synovial membrane mononuclear cells. *Mean, (values range). dRF nephelometry assay, cutoff value >15 UI/ml. All patients with RA had high positive dRF levels (more than three times the ULN for the laboratory and assay): 264 ± 190. eACPA: QUANTA Lite® CCP3.1 IgG/IgA ELISA (INOVA Diagnostics), cutoff value >20 UI/ml. All patients with RA had high positive eACPA levels (more than three times the ULN for the laboratory and assay):125 ± 64. hCRP cutoff value 1 mg/dl. a
b
c
d
e
Penatti et al. Arthritis Research & Therapy (2017) 19:103
Page 3 of 9
joint biopsy and from nine patients with OA and two patients with RA who additionally had knee replacement surgery. The majority of patients with RA had medium/low disease activity (as summarized in Table 1), and were on medium/low doses of disease modifying anti-rheumatic drugs (DMARDs) (hydroxychloroquine, methotrexate or leflunomide). Thirteen out of twenty-four patients were on low-dose corticosteroid therapy ( 0.05 for all. (TIF 642 kb) Additional file 8: Figure S8. Distribution of T helper (a, c) and T regulatory (b, d) subsets in peripheral blood (upper panels) and synovial fluid (lower panels) of significantly different subsets in patients with RA according to CS therapy: statistical analysis by Mann-Whitney test, p > 0.05. Peripheral blood: (a) Th1/17, Th17; (b) Tr1 and TReg subsets. Synovial fluid: (c) Th1/17; (d) TReg subsets. (TIF 607 kb)
Abbreviations ACPA: Anti-citrullinated protein antibody; DAS29: disease activity score in 28 joints; DMARDs: Disease-modifying anti-rheumatic drugs; ELISA: Enzyme-linked immunosorbent assay; ESR: erythrocyte sedimentation rate; HD: Healthy donors; IL: interleukin; NK: Natural killer; NSAIDs: non-steroidal anti-inflammatory drugs; OA: Osteoarthritis; PB: Peripheral blood; PBMCs: Peripheral blood mononuclear cells; RA: Rheumatoid arthritis; RF: Rheumatoid factor; SF: Synovial fluid; SFMCs: Synovial fluid mononuclear cells; SM: Synovial membranes; SMMCs: Synovial membrane mononuclear cells; Th: T helper; Tregs: T regulatory cells; US: Ultrasound Acknowledgements Not applicable. Funding This work was supported by the Cariplo foundation, by the Romeo ed Enrica Invernizzi foundation and by Ricerca Corrente 2015–16 IRCCS Istituto Auxologico Italiano. Availability of data and materials Supporting information for Figures S1, S2, S3, S4, S5, S6, S7 and S8 are available online. Authors’ contributions AP participated in the study design, obtained data, participated in the statistical analysis and drafted the manuscript; FF participated in the study design and coordination, obtained data, participated in the statistical analysis and wrote the manuscript; RDM and PL performed experiments; MP participated in the analysis of the data and in the drafting of the manuscript; AM and ODL recruited patients with OA and RA and obtained biological samples; LP and MT obtained biological samples; SA, MP and AIF participated in the design of the study, in the interpretation of the data and in the drafting of the manuscript; JG and PLM conceived the study, participated in its design and coordination, in analysis and interpretation of data and in the drafting of the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethical approval and consent to participate The ethical committee (Milano Area B) approved the use of blood samples and synovium specimens for research purposes (permission 2476 Comitato Etico Milano Area B/Parere favorevole condizionato datato 02.10.2014/parere 591bis") and informed consent was obtained from all patients. Source of support None declared.
Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 DISCCO-Department of Clinical Science and Community Health Università degli Studi di Milano, 20122 Milan, Italy. 2INGM-National Institute of Molecular Genetics “Romeo ed Enrica Invernizzi”, 20122 Milan, Italy. 3 ASST-Gaetano Pini/CTO Orthopedic and Traumatology Specialist Center, Rheumatology and Orthopedic Department, 20122 Milan, Italy. 4Department of Rheumatology, Leiden University Medical Center, 2300 Leiden, The Netherlands. 5Laboratory of immuno-rheumatological researches, IRCCS Istituto Auxologico Italiano, 20149 Milan, Italy. 6Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20122 Milan, Italy.
Penatti et al. Arthritis Research & Therapy (2017) 19:103
Page 9 of 9
Received: 1 December 2016 Accepted: 2 May 2017
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